Live-cell RESOLFT nanoscopy of transgenic Arabidopsis thaliana

Subdiffraction super-resolution fluorescence microscopy, or nanoscopy, has seen remarkable developments in the last two decades. Yet, for the visualization of plant cells, nanoscopy is still rarely used. In this study, we established RESOLFT nanoscopy on living green plant tissue. Live-cell RESOLFT nanoscopy requires and utilizes comparatively low light doses and intensities to overcome the diffraction barrier. We generated a transgenic Arabidopsis thaliana plant line expressing the reversibly switchable fluorescent protein rsEGFP2 fused to the mammalian microtubule-associated protein 4 (MAP4) in order to ubiquitously label the microtubule cytoskeleton. We demonstrate the use of RESOLFT nanoscopy for extended time-lapse imaging of cortical microtubules in Arabidopsis leaf discs. By combining our approach with fluorescence lifetime gating, we were able to acquire live-cell RESOLFT images even close to chloroplasts, which exhibit very strong autofluorescence. The data demonstrate the feasibility of subdiffraction resolution imaging in transgenic plant material with minimal requirements for sample preparation.